A multifaceted cellular damage repair and prevention pathway promotes high level tolerance to β-lactam antibiotics

Abstract

18Bactericidal antibiotics are powerful drugs due to their ability to not only inhibit essential 19 bacterial functions, but to convert them into toxic (and potentially lethal) processes. 20However, many important bacterial pathogens are remarkably tolerant against 21 bactericidal drugs, due to inducible stress responses that repair antibiotic-induced 22 damage. The mechanistic details of how stress responses promote whole population 23 tolerance in important human pathogens are unknown. The two-component system 24 VxrAB of the diarrheal pathogen Vibrio cholerae, a model system for high-level -lactam 25 2 tolerance, is induced by exposure to cell wall acting antibiotic and controls a gene 26 network encoding highly diverse functions, including cell wall synthesis functions and 27 iron uptake systems. Here, we show that positive control over cell wall synthesis 28 functions only partially explains high level -lactam tolerance. We find that in addition to 29 cell wall damage, -lactam antibiotics inappropriately induce the Fur-regulated iron 30 starvation response, causing an increase in intracellular free iron levels and colateral 31 oxidative damage. We propose that VxrAB reduces antibiotic-induced toxic influx of 32 Fe 2+ and concomitant metabolic perturbations by selectively downregulating iron uptake 33 transporters. Our results suggest that the ability to counteract diverse antibiotic-induced 34 stresses promotes high-level antibiotic tolerance and highlight the complex responses 35 elicited by antibiotics in addition to their primary mechanism of action. 36 37 by either developing the ability to grow in their presence (antibiotic resistance, ABR) or 49 to simply stay alive in their presence for extended time periods (antibiotic 50 tolerance/persistence) [3][4][5][6][7][8] . While the mechanisms and consequences of ABR are 51 relatively well-established, antibiotic tolerance remains poorly understood, limiting our 52 ability to develop antibiotic adjuvants that increase the efficacy of existing drugs. 53 54The -lactam antibiotics (penicillins, cephalosporins, carbapenems, cephamycins and 55 monobactams) are highly potent bactericidal agents. Their typically lethal action results 56 from their ability to simultaneously inhibit multiple targets (i.e, the transpeptidase 57 domain of multiple penicillin-binding proteins [PBPs]), which ultimately causes bacterial 58 cells to deplete essential cell wall precursors and self-destruct through the activity of 59 endogenous, cell wall lytic enzymes ('autolysins'; endopeptidases, amidases and lytic 60 transglycosylases) 9-12 . However, we and others have recently shown that many 61 clinically significant Gram-negative pathogens are remarkably -lactam tolerant. The 62 cholera pathogen Vibrio cholerae, the opportunistic pathogen Pseudomonas aeruginosa 63 and clinical isolates of Enterobacteriaceae all survive treatment with -lactam antibiotics 64 (including the "last resort" agent meropenem) by forming non-dividing, cell wall deficient 65 spheroplasts 11,13,14 . Upon...

Understanding tolerance to cell wall–active antibiotics

“…Intriguingly, V. cholerae accumulates high intracellular iron levels upon β-lactam exposure and this is exacerbated in the vxrAB mutant. 128 Further, β-lactam exposure resulted in induction of the Fur iron starvation response, likely due to direct oxidation of its Fe 2+ corepressor by β-lactam-induced H 2 O 2 production. 128 It thus appears that VxrAB's role in tolerance is at least partially to tone down an out-of-control iron starvation response that would otherwise overload β-lactam-stressed cells with iron, causing downstream toxic events like the generation of ROS.…”

“…128 Further, β-lactam exposure resulted in induction of the Fur iron starvation response, likely due to direct oxidation of its Fe 2+ corepressor by β-lactam-induced H 2 O 2 production. 128 It thus appears that VxrAB's role in tolerance is at least partially to tone down an out-of-control iron starvation response that would otherwise overload β-lactam-stressed cells with iron, causing downstream toxic events like the generation of ROS. Consistent with this model, deleting iron transporters partially restored high tolerance levels to a vxrAB mutant.…”

“…Thus, not surprisingly, exposure to such agents results in various sequelae that reflect distinct cellular responses as well as purely physical consequences of massive cell wall damage. Exposure to β-lactams has been tied to the generation of reactive oxygen species (ROS), 125 or not; 126,127 activation of iron uptake; 128 changes in TCA cycle intermediate concentrations; 129 induction of the heat shock response; 130,131 induction of the SOS DNA damage response; 132,133 RNA degradation; 116,134 and stringent response (SR) induction. 135 In principle, any or all of these processes may contribute to some degree to β-lactam-mediated cell death (or tolerance, as shall be considered later), but their detailed contributions have remained poorly understood.…”

“…Consistent with this model, deleting iron transporters partially restored high tolerance levels to a vxrAB mutant. 128 The VxrAB system thus aptly illustrates the complex physiological consequences of β-lactam exposure and tolerance pathways, where cells not only have to overcome cell wall loss, but also remediate toxic iron influx and potentially downstream accumulation of toxic ROS.…”

Antibiotic tolerance

“…Similar to L-forms, the activity of cell wall lytic enzymes is required for spheroplast formation [ 20 ]. Recovery from the spheroplast state requires cell envelope stress responses, cell wall synthesis functions, and likely, a reduction in the formation of ROS [ 23 ]. The key difference between spheroplasts and L-forms is that the former do not divide.…”